Abstract
The site-specific recombinase Tn3 resolvase initiates DNA strand exchange when two res recombination sites and six resolvase dimers interact to form a synapse. The detailed architecture of this intricate recombination machine remains unclear. We have clarified which of the potential dimer-dimer interactions are required for synapsis and recombination, using a novel complementation strategy that exploits a previously uncharacterized resolvase from Bartonella bacilliformis ("Bart"). Tn3 and Bart resolvases recognize different DNA motifs, via diverged C-terminal domains (CTDs). They also differ substantially at N-terminal domain (NTD) surfaces involved in dimerization and synapse assembly. We designed NTD-CTD hybrid proteins, and hybrid res sites containing both Tn3 and Bart dimer binding sites. Using these components in in vivo assays, we demonstrate that productive synapsis requires a specific "R" interface involving resolvase NTDs at all three dimer-binding sites in res. Synapses containing mixtures of wild-type Tn3 and Bart resolvase NTD dimers are recombination-defective, but activity can be restored by replacing patches of Tn3 resolvase R interface residues with Bart residues, or vice versa. We conclude that the Tn3/Bart family synapse is assembled exclusively by R interactions between resolvase dimers, except for the one special dimer-dimer interaction required for catalysis.
Highlights
The resolvases of the bacterial transposons Tn3 and γδ are closely related DNA site-specific recombinases and founder members of the serine recombinase family (Rowland and Stark, 2005; Stark, 2014)
We report the design and testing of a modular synthetic res site, which can be tailored to support either Tn3 or Bart resolvase-mediated recombination by changing the DNA sequence motifs recognized by the C-terminal domains (CTDs)
The two key innovations that allowed us to reach these conclusions were our characterization of the Bart resolvase recombination system and our “patch mutagenesis” strategy to remodel the R interface
Summary
The resolvases of the bacterial transposons Tn3 and γδ are closely related DNA site-specific recombinases and founder members of the serine recombinase family (Rowland and Stark, 2005; Stark, 2014). Some of these hybrid res substrates were fully or substantially resolved when just a single resolvase with Bart CTDs (B–B or T–B) was expressed (lines 1 and 2) This suggests that the Bart CTD can bind weakly to t sites, aided by the extensive cooperative NTD interactions within the synapse. Some early models of the synapse (Krasnow et al, 1983; Boocock et al, 1986; Sarkis et al, 2001; Rowland et al, 2002) proposed that dimers bound at the res accessory sites (II and III) might mediate synaptic interactions via the “catalytic” dimer–dimer interface (as at site I) or another type of NTD–NTD interface To test these proposals, we used the substrates tbb × ttt and btt × ttt (Figure 6c). Apart from the well-characterized catalytic tetramer-forming interactions at site I of res, the productive synapse is assembled using only R interactions between resolvase dimers
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